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<i>Sagittaria trifolia</i> is a medicinal foodstuff of China and East Asia belonging to the family Alismataceae. Samples of <i>S. trifolia</i> tubers were collected from Meihekow, Siping, Jilin, Harbin and Wuchang from Northeast China. The current study was aimed to evaluate the qualitative and quantitative analysis, antioxidant activity, biochemical analysis and chemical composition of different populations of <i>S. trifolia.</i> By using Folin&#8722;Ciocalteu, aluminium chloride colourimetric and 1,1-diphenyl-1-picrylhydrazyl (DPPH), total phenol and flavonoids content and antioxidant activity was analysed. Furthermore, chemical composition, biochemical analysis and mineral substances were also determined. The results showed the presence of flavonoids, phenols, saponins, tannins, glycosides and steroids except for alkaloids and terpenoids by qualitative analysis. Quantitative analysis revealed that highest total phenol, flavonoids content and antioxidant potential identified from Meihekow, i.e., 2.307 mg GAE/g, 12.263 mg QE/g and 77.373%, respectively. Gas chromatography-mass spectrometry results showed the presence of 40 chemical compounds corresponding to 99.44% of total extract that might be responsible for antioxidant properties. Mineral and biochemical analysis revealed the presence of calcium, magnesium, potassium, sodium, iron, copper, zinc and, carbohydrate, protein, fibre and fat contents, respectively. Interestingly, all <i>S. trifolia</i> populations collected from different locations possess similar composition. The dietary values, phytoconstituents, antioxidant activities and nutritional and curative chemical compounds of <i>S. trifolia</i> are beneficial for the nutritherapy of human beings.